Most proteins and enzymes are composed of subunits held together by non-covalent bonds. This particular construction offers advantages to the living organism particularly with respect to control of cellular processes. In order to understand, at the molecular level, regulatory processes it is therefore important to define the subunit interactions of oligomeric enzymes. We plan to investigate the quaternary structure of proteins utilizing two novel approaches which should be generally applicable to oligomeric systems. The first approach involves the rapid determination of the equilibrium constants for the association of protein subunits by concentration difference spectra. The method we have developed requires no knowledge of the extinction coefficients of the oligomer or monomer or changes in extinction coefficients upon polymerization. The second approach involves the determination of the blocks of amino acids from the primary structure of a protein or enzyme which are involved at the subunit contact sites. These sequences will be determined by isolation of peptides which comprise the subunit contact site by limited proteolysis of the protein. We shall also use immunological methods of producing antibodies which should be specific for the subunit contact sites of the proteins. Our methods will be tested upon two proteins: glyceraldehyde- phosphate dehydrogenase and hemerythrin.